Preliminary report of de novo adipogenesis using novel bioabsorbable implants and image evaluation using a porcine model.

Our bioabsorbable poly-L-lactic acid (PLLA) mesh implants containing collagen sponge are replaced with adipose tissue after implantation, and this is an innovative method for breast reconstruction. In this preliminary study, we investigated the formation of adipose tissue and evaluated the process via multimodal images in a porcine model using an implant aggregate to generate the larger adipose tissue. The implant aggregate consists of PLLA mesh implants containing collagen sponge and a poly-glycolic acid woven bag covering them. We inserted the implant aggregates under the porcine mammary glands. Magnetic resonance imaging (MRI), ultrasonography (USG), and 3-dimensional (3D) surface imaging and histological evaluations were performed to evaluate the formation of adipose tissue over time. The volume of the implant aggregate and the formed adipose tissue inside the implant aggregate could be evaluated over time via MRI. The space within the implant aggregate was not confirmed on USG due to the acoustic shadow of the PLLA threads. The change in volume was not confirmed precisely using 3D surface imaging. Histologically, the newly formed adipose tissue was confirmed on the skin side of the implant aggregate. This implant aggregate has the ability to regenerate adipose tissue, and MRI is an appropriate method for the evaluation of the volume of the implant aggregation and the formation of adipose tissue.

Small-Diameter Synthetic Vascular Graft Immobilized with the REDV Peptide Reduces Early-Stage Fibrin Clot Deposition and Results in Graft Patency in Rats.

Early-stage hemocompatibility is indispensable for manufacturing tissue-engineered vascular grafts used in regenerative medicine. In this study, we report the in vivo blood response and patency of small-diameter synthetic vascular grafts modified with the Arg-Glu-Asp-Val (REDV) peptide. Vascular grafts were prepared by casting REDV-conjugated poly(depsipeptide-co-caprolactone) on a stainless-steel mandril (diameter: 1.8 mm). After implanting the grafts into the abdominal aorta of rats for 24 h, all three control grafts without the peptide and three out of the four REDV (control sequence) peptide-modified grafts showed occlusion. The luminal surfaces of these grafts were covered with thick thrombi. In contrast, all the grafts containing the REDV peptide were patent, and their luminal surfaces were covered with a thin layer of fibrin. These results indicated that the REDV peptide on the luminal surface effectively reduced early-stage fibrin clot deposition and formed the pseudo-endothelium layer in a peptide sequence-specific manner, resulting in graft patency.

Circulating endothelial progenitor cells in small-diameter artificial blood vessel.

In the tissue engineering research field, the presence of circulating endothelial progenitor cells (EPCs) in the peripheral blood of adults represents a promising cell source to grow autologous endothelium on blood-contacting devices. Materials functionalized with EPC-specific molecules are an intriguing strategy to induce the homing and differentiation of the trapped EPCs into endothelial cells to generate a non-thrombogenic surface. Although the EPCs have been identified in adult peripheral blood about 30 years ago, in the subsequent literatures, the term "EPCs" has encompassed different cell populations with a mixed ability to contribute to the formation of blood vessels. This confusion is due to limited functional characterization of "EPCs", an improper nomenclature, and the poorly standardized protocols. This review will screen the literature about "EPCs" to propose a specific nomenclature, phenotypic characterization, and, eventually, a protocol to perform reliable experiments.

Decellularization of submillimeter-diameter vascular scaffolds using peracetic acid.

Various decellularization methods for allogenic and xenogenic bioscaffolds have been previously reported; however, decellularization methods for very thin (submillimeter-diameter) vascular tissues have not been discussed well. In this study, rat tail arteries (inner diameter, 0.6 mm) were decellularized with peracetic acid (PAA) and DNase I. PAA treatment is expected not only to disrupt cell membranes which improves the decellularization efficiency in the subsequent DNase treatment, but also to sterilize vascular scaffolds. We succeeded in adequate cell removal by immersing in 0.3% isotonic PAA solution and subsequent washing with DNase solution. For the DNase washing process, the perfusion method was superior in terms of cell removal to the static immersion method. Graft lumen was modified with a peptide composed of a collagen binding sequence and endothelial progenitor cell-binding sequence, (Pro-Hyp-Gly)7-Gly-Gly-Gly-Arg-Glu-Asp-Val, as previously reported. They were patent in rat allogeneic transplantation model for 2 weeks, but unexpectedly resulted in graft rupture or tear formation, thereafter, suggesting reduced mechanical strength of the decellularized scaffolds. Histology showed that the thickness of the extracellular matrix (ECM) was decreased by the perfusion of the DNase solution. The method of combination of PAA and DNase was not necessarily optimal for the decellularization of very thin vascular tissues. The decellularization method is a compromise between effective cell removal and maintenance of the ECM nature. Since the acceptability of ECM denaturation by the host tissue highly depends on individual cases, decellularization methods should be carefully selected according to the type of target tissue and its intended use.

Simple and efficient method for consecutive inactivation-cryopreservation of porcine skin grafts.

We previously reported that inactivation treatment by high hydrostatic pressurization (HHP) has potential utility as a novel skin regeneration therapy for various skin tumors. In this study, we evaluated whether glycerol-cryopreservation could be applied in order to preserve inactivated skin by HHP using a porcine model. Twenty full-thickness skin grafts (1.5 × 1.5 cm) were prepared from a minipig. The skin samples were inactivated by the HHP in normal saline or glycerol/fructose solution, followed by cryopreservation for 5 weeks at - 80 °C in each same solution. Another 10 grafts immediately after inactivation were prepared as non-cryopreserved controls. Nine grafts in each group were randomly implanted on the fascia of a host pig and removed at 1, 4 and 11 weeks after grafting. All grafts showed engraftment macroscopically. Hematoxylin eosin staining showed the cellular components in all areas of the dermis at 4 and 11 weeks after grafting, and immunohistochemical staining for CD31 showed the presence of capillaries in the grafts in all groups. The surface and cross-sectional areas of grafts in the normal saline solution cryopreserved group decreased between 1 and 11 weeks, whereas these areas in the glycerol cryopreserved group did not decrease significantly. Glycerol cryopreservation may therefore be a simple and efficient method for preserving porcine skin inactivated by HHP.

Design of in situ porcine closed-circuit system for assessing blood-contacting biomaterials.

The overall pre-clinical process of determining the blood compatibility of any medical device involves several stages. Although the primary purpose is to protect the patients, laboratory testing has been over-utilized for many years with a huge number of unnecessary animal tests being done routinely. Recently, the elimination of needless testing has become important in controlling the cost of healthcare and in addressing many issues related to the ethics of animal research. With this in mind, we designed a new in situ porcine closed-circuit system to study the complex interplay between platelets, coagulation proteins, and other cellular elements in pigs. We proved that this system can be implemented in blood compatibility testing and minimize the number of animals used in the experiments.

Periodontal regeneration induced by porous alpha-tricalcium phosphate with immobilized basic fibroblast growth factor in a canine model of 2-wall periodontal defects.

We evaluated the effect of porous alpha-tricalcium phosphate (α-TCP) with immobilized basic fibroblast growth factor (bFGF) on periodontal regeneration in a canine model of 2-wall periodontal defects. Identical bone defects were made in the canine mandible; six defects in each animal were filled with porous α-TCP with bFGF bound via heparin (bFGF group), and the remaining defects were filled with unmodified porous α-TCP (control group). Micro-computed tomography and histological evaluation were performed at 2, 4, and 8 weeks post-implantation. The bone mineral content of the bFGF group was higher than that of the control group at 2 and 4 weeks (p < 0.05). Histological evaluation at 2 weeks post-implantation revealed degradation of the porous α-TCP, and bone had formed on the surface of α-TCP particles in the bFGF group. Some of these collagen fibers connected the newly formed cementum with the alveolar bone, revealing the formation of new periodontal ligaments with Sharpey's fibers. At 8 weeks, continuous cortical bone with a Haversian structure covered the top of the bone defects in the bFGF group. These findings indicate that porous α-TCP with immobilized bFGF could promote periodontal regeneration at the early regeneration phase in a canine model of 2-wall periodontal defects.

Label-Free Separation of Induced Pluripotent Stem Cells with Anti-SSEA-1 Antibody Immobilized Microfluidic Channel.

When induced pluripotent stem cells (iPSCs) are routinely cultured, the obtained cells are a heterogeneous mixture, including feeder cells and partially differentiated cells. Therefore, a purification process is required to use them in a clinical stage. We described a label-free separation of iPSCs using a microfluidic channel. Antibodies against stage-specific embryonic antigen 1 (SSEA-1) was covalently immobilized on the channel coated with a phospholipid polymer. After injection of the heterogeneous cell suspension containing iPSCs, the velocity of cell movement under a liquid flow condition was measured. The mean velocity of the cell movement was 2.1 mm/sec in the unmodified channel, while that in the channel with the immobilized-antibody was 0.4 mm/sec. The eluted cells were fractionated by eluting time. As a result, the SSEA-1 positive iPSCs were mainly contained in later fractions, and the proportion of iPSCs was increased from 43% to 82% as a comparison with the initial cell suspension. These results indicated that iPSCs were selectively separated by the microfluidic channel. This channel is a promising device for label-free separation of iPSCs based on their pluripotent state.

Hepatocytic differentiation of iPS cells on decellularized liver tissue.

Decellularized tissues (DETs) have been attracting great attention as scaffolds for tissue-engineering approaches. Recently, some studies have reported that decellularized liver tissues (DLT) can provide an excellent environment for the hepatocytic differentiation of hepatic stem/progenitor cells that were already committed to the hepatocyte lineage. However, the effects of DLT on the hepatocytic differentiation of induced pluripotent stem cells (iPSs) have not yet been established. Here we studied the hepatocytic differentiation of iPSs on DLT and decellularized heart tissues (DHT) in order to determine the tissue-specific effects of DETs on iPSs differentiation. Our results showed that DLTs led to higher gene expression levels of forkhead box A2 (a marker of endoderm) and CCAAT/enhancer binding protein-α (master transcription factor to hepatocyte differentiation), alpha-fetoprotein (a marker of fetal hepatocyte,), and albumin (a marker of fetal and mature hepatocyte) of iPSs than on DHTs. Furthermore, gene expression levels of tyrosine aminotransferase (a marker of mature hepatocyte) were higher on DLT than that on DHT, and immunocytochemical analysis and ELISA assay showed that albumin secretion level of iPSs on DLT was higher than that on DHT. Our study demonstrated that the use of DLTs led to mature hepatocytic differentiation levels of iPSs compared to DHTs, which provides a better niche for iPSs cell engineering and enables the preparation of useful mature cells for regenerative therapy.

A Novel Strategy for Etiologic Factor Removal: Drug-Navigated Clearance System (DNCS).

Here, we propose a novel therapeutic concept named drug-navigated clearance system (DNCS), in which a "navigator" decreases the concentration of a target etiologic factor in the blood by steering it to an unusual metabolic pathway. The navigator is composed of protein A (ProA) and dextran sulfate (DexS) and it successfully navigated antibodies (ABs), a model etiologic factor of dilated cardiomyopathy, to hepatocytes in vitro in the presence of low-density lipoprotein (LDL). ProA captured the Fc region of the target antibody while the DexS bound to LDL via the well-known electrostatic interaction. The hepatocytes simultaneously took up LDL via the LDL-receptor and internalized the AB/ProA-DexS complex that was bound to LDL. Therefore, this process demonstrates our attempt to navigate the etiologic factor to an alternate target pathway such as the LDL salvage.

Airway reconstruction using decellularized tracheal allografts in a porcine model.

PURPOSE: Tracheal cartilage reconstruction is an essential approach for the treatment of tracheal congenital abnormalities or injury. Here, we evaluated the use of allogeneic decellularized tracheas as novel support scaffolds. METHODS: Six weaned pigs (4-week-old domestic males) were transplanted with allogeneic tracheal graft patches (three decellularized and three fresh tracheal scaffolds) onto artificial defects (approximately 15 × 15 mm). After 11 weeks, the tracheas were evaluated by bronchoscopy and histological studies. RESULTS: No pigs displayed airway symptoms during the observation period. Tracheal lumen restored by fresh graft patches showed more advanced narrowing than that treated with decellularized grafts by bronchoscopy. Histologically, fresh grafts induced typical cellular rejection; this was decreased with decellularized grafts. In addition, immunohistochemistry demonstrated regenerating foci of recipient cartilage along the adjacent surface of decellularized tracheal grafts. CONCLUSION: Decellularized allogeneic tracheal scaffolds could be effective materials for restoring impaired trachea.

Verification of the Inactivation of Melanocytic Nevus in vitro Using a Newly Developed Portable High Hydrostatic Pressure Device.

High hydrostatic pressure (HHP) technology is a physical method for inactivating tissue. We reported that nevus specimens were inactivated after HHP at 200 MPa and that the inactivated nevus could be used as autologous dermis for covering skin defects. In this study, we verified the inactivation of nevus specimens using a newly developed portable HHP device which will be used in a clinical trial. Nevus tissue specimens were obtained from 5 patients (mean age 7.2 years, range 1-19). We cultured fibroblasts and nevus cells from the tissue specimens and then evaluated their inactivation after HHP at 200 MPa by confirming the attachment of the suspensions and by the live/dead staining of the suspensions, through the dissociation of the cells on chamber slides and by the live/dead staining of the remaining cells. The cells were also quantitatively evaluated by WST-8 assay. We then confirmed the inactivation of the nevus specimens after HHP using explant culture. Our results indicated that fibroblasts and nevus cells were inactivated after HHP at 200 MPa, with the exception of a small percentage of green-colored cells, which reflected the remaining activity of the cellular esterases after HHP. No cells migrated from the nevus specimens after HHP at 200 MPa. We verified the inactivation of fibroblasts and nevus cells cultured from nevus specimens, and in the nevus samples themselves after pressurization at 200 MPa using this device. This device could be used in clinical trials for giant congenital melanocytic nevi and may thus become useful in various medical fields.

Single-step immobilization of cell adhesive peptides on a variety of biomaterial substrates via tyrosine oxidation with copper catalyst and hydrogen peroxide.

Immobilization of biologically active peptides which were isolated from extracellular matrix proteins is a powerful strategy for the design and functionalization of biomaterial substrates. However, the method of peptide immobilization was restricted, that is, peptide is often immobilized through the reactive groups inherent in substrates with multistep reactions. Here, we report a single-step immobilization of fibronectin-derived cell adhesive peptide (Arg-Glu-Asp-Val; REDV) onto polymer materials by use of tyrosine oxidation with copper catalyst and hydrogen peroxide. REDV peptide was successfully immobilized on tissue culture polystyrene, poly(ethylene terephthalate), poly(vinyl chloride), expanded-poly(tetrafluoroethylene), and poly(l-lactic acid), resulting in enhanced adhesion of human umbilical vein endothelial cells. This method is a single-step reaction under very mild conditions and is available for the biological functionalization of various medical devices.

Micro-CT evaluation of high pressure-decellularized cardiovascular tissues transplanted in rat subcutaneous accelerated-calcification model.

We have succeeded in reducing the calcification of acellular aortas or valves in porcine allogeneic system by removing the DNA and phospholipids, but its further reduction is desirable. Here, the calcification of the acellular tissue was evaluated in rat subcutaneous transplantation model which is known as calcification model. Acellular samples prepared by high-hydrostatic pressure (HHP) protocols with different washing media were implanted and the calcification was monitored under micro-computed tomography for 1 and 3 months. The amount of the calcium deposition was quantitatively evaluated by atomic absorption spectroscopy. A cell culture medium showed very good cell removal ability but led to severe calcification at 1 month, and surprisingly the calcium deposition increased as the washing period increased. This calcification was suppressed by removing the DNA fraction with high DNase concentration. On the other hand, the calcification was greatly reduced when washed with saline even at low DNase concentration after 2 weeks washing. These results suggest that the ion species in the washing medium and the residual DNase cooperatively affect the tendency of in vivo calcification, which led us to the possibility of reduced calcification of acellular cardiac tissues.

Long-term/bioinert labeling of rat mesenchymal stem cells with PVA-Gd conjugates and MRI monitoring of the labeled cell survival after intramuscular transplantation.

Noninvasive in vivo imaging of transplanted stem cells is an effective method to clarify the mechanisms involved in stem cell transplantation therapy. We labeled rat mesenchymal stem cells (MSCs) with water-soluble magnetic resonance imaging (MRI) contrast agent poly(vinyl alcohol)-gadolinium (PVA-Gd) in order to ascertain the fate of transplanted MSCs in vivo. PVA-Gd was retained and localized in the cytosolic compartment of MSCs for a longer period of time. The effect of PVA-Gd labeling on MSC proliferation was much less than that of the commercially available contrast agent ProHance, and the labeled MSCs were found to have osteoblastic differentiation ability. To study the MSC lifetime in vivo, MSCs were seeded and trapped in the cytocompatible three-dimensional porous scaffolds of Spongel and transplanted. The MRI signal attributed to MSCs was eliminated from the transplanted site in 14 days. Because free PVA-Gd was rapidly eliminated from the site, this signal reduction indicated MSC death in the transplantation site. The low efficiency of MSC transplantation for ischemic tissue may be due to their short lifetime, making it important to develop highly effective stem cell transplantation systems that address cell number, injection position, and cell formulation (suspension, sheet, and aggregates). Our cell survival tracking system would be a very powerful tool to this end and would be applicable in clinical cell therapies.

Reduction of inorganic phosphate-induced human smooth muscle cells calcification by inhibition of protein kinase A and p38 mitogen-activated protein kinase.

High levels of serum phosphate are associated with calcification of human smooth muscle cells (HSMCs). We investigated whether inhibition of protein kinase A (PKA) and mitogen-activated protein kinase (MAPK) signals [p38, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK)] can reduce inorganic phosphate (Pi)-induced HSMC calcification. Inhibition of PKA or p38 MAPK by inhibitors or small interfering RNAs (siRNAs) reduced Ca levels and alkaline phosphatase activities in HSMCs treated with high Pi, but inhibition of ERK1/2 and JNK showed no significant changes. Moreover, there were no significant changes in cell viability on adding siRNAs and three inhibitors (PKA, p38, and MEK1/2), but JNK inhibitor slightly reduced cell viability. These results show that PKA and p38 MAPK are involved in the Pi-induced calcification of HSMCs, and may be good targets for reducing vascular calcification.

Water absorbing and quick degradable PLLA/PEG multiblock copolymers reduce the encapsulation and inflammatory cytokine production.

Biomaterials that contact with soft tissues such as postoperative adhesion prevention membrane or tissue-regenerative scaffolds should possess specific features such as hydrophilicity, mild to no immunogenicity, and quick degradability. The inflammation reaction to multiblock copolymers of poly(L-lactic acid) (PLLA) and poly(ethylene glycol), named as Multi, which we developed as a good adhesion prevention materials with a very high degradation rate were investigated and compared with usual PLLA, non-degradable polyethylene (PE), and acellular collagenous tissue (COL). Tissue encapsulation, inflammatory cell recruitment, and expression of four cytokines (IL-1ß, IL-6, IL-10, and TGFß) affecting the promotion or inhibition of inflammation and wound healing were evaluated. The thick encapsulation for PE might have related to high expression of TGFß, and it was largely reduced in the cases of PLLA and Multi. The cytokine expression pattern in PE was dominantly alternatively activated macrophage (M2) type, while expression patterns to Multi were classically activated macrophage (M1)-type dominant, as with the COL specimen. Thus, multi is a tissue compatible material in spite of the large degradability. By introducing low molecular weight PEG into PLLA as multiblock-type sequence, we successfully prepared biocompatible PLLA derivatives with high molecular weight, large degradation rate, and mild tissue responses.

Intima/medulla reconstruction and vascular contraction-relaxation recovery for acellular small diameter vessels prepared by hyperosmotic electrolyte solution treatment.

This study aims at the evaluation of blood vessel reconstruction process of decellularized small diameter vessels prepared by a hyperosmotic electrolyte solution treatment not only histologically but also physiologically in rat transplantation model. Complete cell removal by a hyperosmotic electrolyte solution treatment was confirmed by hematoxylin/eosin staining and scanning electron microscopic observation. All acellular vessels transplanted into the rat abdominal aorta were patent up to 14 months. One week post-transplantation, the vWF-positive cells were observed on the luminal surface but the layer formation did not complete. Five weeks following transplantation, the vWF-positive endothelial cells were located on the intima consistent with intact endothelial cells. Beneath the endothelial cells, α-SMA-positive smooth muscle cells were distributed. The harvested vessels displayed formation of tunica intima (endothelial cells) and tunica medulla (smooth muscle cell) layers. We also examined the physiological properties of the vessels 12 months post-transplantation using a wire myograph system. The transplanted vessels contracted upon addition of norepinephrine and relaxed upon addition of sodium nitroprusside as well as the native vessels. In conclusion, the acellular vessels prepared with hyperosmotic electrolytic solution showed excellent and long-term patency, which may be related to the successful preservation of vascular ECM. In addition, the acellular vessels revealed the intima/medulla regeneration with the physiological contraction-relaxation functions in response to the each substance.

High pressure pasteurization: Simultaneous native tissue decellularization and sterilization.

Introduction: Terminal sterilization is important for the clinical applicability of decellularized xenografts. High hydrostatic pressurization (HHP) process is a potential strategy for decellularization and decontamination of xenografts; however, its disinfection efficiency remains poorly elucidated. This study investigated the disinfection efficacy of the HHP process at physiologically relevant 36 °C against difficult-to-kill spore-forming bacteria. Methods: Bacillus atrophaeus and Geobacillus stearothermophilus were suspended in a pressurization medium with or without antibiotic agents and pressurized under two different HHP procedures: repeated and sustained pressurization. Results: The sustained pressurizing conditions, exploited for the conventional tissue decellularization, did not effectively eliminate the bacteria; however, repeated pressurization greatly increased the disinfection effect. Moreover, the antibiotic-containing pressurization medium further increased the disinfection efficiency to the level required for sterilization. Conclusions: The optimized high hydrostatic pressurization can be used to sterilize biological tissues during the decellularization process and is a promising strategy for manufacturing tissue-derived healthcare products.